Podcasts about Mutagenesis

Hear NEB Senior Tech Support Scientist Rachel Carver-Brown explain site-directed mutagenesis and multi-site mutagenesis techniques.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534746v1?rss=1 Authors: Kettel, P., Marosits, L., Spinetti, E., Rechberger, M., Radler, P., Niedermoser, I., Fischer, I., Versteeg, G. A., Loose, M., Covino, R., Karagoz, G. E. Abstract: Upon accumulation of unfolded proteins at the endoplasmic reticulum (ER), IRE1 activates the unfolded protein response (UPR) to restore protein-folding homeostasis. During ER stress, the ER lumenal domain (LD) of IRE1 drives its clustering on the ER membrane to initiate signaling. How IRE1 LD assembles into high-order oligomers remains largely unknown. By in vitro reconstitution experiments we show that human IRE1 LD forms dynamic biomolecular condensates. IRE1 LD condensates were stabilized when IRE1 LD was tethered to model membranes and upon binding of unfolded polypeptide ligands. Molecular dynamics simulations suggested that weak multivalent interactions are involved in IRE1 LD assemblies. Mutagenesis showed that disordered regions in IRE1 LD control its clustering in vitro and in cells. Importantly, dysregulated clustering led to defects in IRE1 signaling. Our results reveal that membranes and unfolded polypeptides act as scaffolds to assemble dynamic IRE1 condensates into stable, signaling competent clusters. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Tim Fenton, Ph.D., University of Southampton, shares his work on the roles of APOBEC3 genes. Hear how he is investigating APOBEC regulation and function in keratinocytes. Series: "Stem Cell Channel" [Health and Medicine] [Show ID: 38724]

Tim Fenton, Ph.D., University of Southampton, shares his work on the roles of APOBEC3 genes. Hear how he is investigating APOBEC regulation and function in keratinocytes. Series: "Stem Cell Channel" [Health and Medicine] [Show ID: 38724]

Tim Fenton, Ph.D., University of Southampton, shares his work on the roles of APOBEC3 genes. Hear how he is investigating APOBEC regulation and function in keratinocytes. Series: "Stem Cell Channel" [Health and Medicine] [Show ID: 38724]

Tim Fenton, Ph.D., University of Southampton, shares his work on the roles of APOBEC3 genes. Hear how he is investigating APOBEC regulation and function in keratinocytes. Series: "Stem Cell Channel" [Health and Medicine] [Show ID: 38724]

Tim Fenton, Ph.D., University of Southampton, shares his work on the roles of APOBEC3 genes. Hear how he is investigating APOBEC regulation and function in keratinocytes. Series: "Stem Cell Channel" [Health and Medicine] [Show ID: 38724]

Tim Fenton, Ph.D., University of Southampton, shares his work on the roles of APOBEC3 genes. Hear how he is investigating APOBEC regulation and function in keratinocytes. Series: "Stem Cell Channel" [Health and Medicine] [Show ID: 38724]

Tim Fenton, Ph.D., University of Southampton, shares his work on the roles of APOBEC3 genes. Hear how he is investigating APOBEC regulation and function in keratinocytes. Series: "Stem Cell Channel" [Health and Medicine] [Show ID: 38724]

I read my great, lost essay "A Mutagenesis" from Generation Hex, in which I give a grand overview theory of Magick as the missing key to genetic engineering. Yes, it is as wacky as it sounds, and yes, you are going to love it. Check it! PS. Micki Pellerano's ASTROLOGY OF WEALTH is now out and it is AWESOME! Learn how to harnass the power of planetary magick and astrology to supercharge your wealth and career success. Open now! See it at: https://www.magick.me/p/astrology

Join the Weeknight Hero team once against with the finale of the Thursday Night in the Danger Room arc. With the session from the previous episode resulting in a completely destroyed danger room, Madame Pele must direct her students to safety before the entire building falls down. 

Join us once again for the continuation of the Mutagenesis solo series as Madame Pele puts her mutant students through a final combat exercise to demonstrate their teamwork and her leadership abilities. Katie's ability to lead and teach will decide the fates of her charges as they do battle with her greatest obstacle yet, the Dynamo Buster.

Join us for the continuation of the Mutagenesis solo series as Madame Pele presents her students with a final training program to put their newly understood powers to the test. Katie will decide who will succeed and who will fail based on her leadership skills.

  Vidcast:  https://youtu.be/wrNvC96y3a4   The media has been heralding the development of molnupiravir, an oral anti-viral agent developed by Merck and Ridgeback Biotherapeutics, that reduced the incidence of hospitalization and death for CoVid patients by about 50%.  Let's look at the fine print about this drug.     The 50% number comes from a Merck phase 3 randomized, controlled, double-blind multi-site global trial of molnupiravir which was stopped at the 90% recruitment level due to “compelling results.” Those participating had positive CoVid PCR tests, mild-to-moderate infections, at least one complicating risk factor such as obesity, diabetes, or older age and began the drug within 5 days of symptom onset.   This orally administered drug inhibits viral replication by causing local mutations in the viral RNA.  While those in the study did not have any worrisome short-term adverse effects, details of side effects of the drug have not been released and there is the possibility that molnupiravir could mutate a patient's DNA with catastrophic results.  For this reason, those in the trial were warned to abstain from sex and to use contraception.   This drug was previously tried in hospitalized CoVid patients without a significant clinical benefit.  It does seem to work earlier in the infection cycle, but there may well be a limited number of days after infection during which it will benefit a patient.  And when it is of use, there is only a 50-50 chance you will be helped.   This drug may turn out to be safe and useful, but for now buyer beware.  If it is proven useful, it will be added to drugs such as monoclonal antibody cocktails and Remdesivir for early disease and dexamethasone and tocilizumab for later disease.  The best defense, though,  is prevention with a CoVid vaccine…. so get vaccinated.   https://www.merck.com/news/merck-and-ridgebacks-investigational-oral-antiviral-molnupiravir-reduced-the-risk-of-hospitalization-or-death-by-approximately-50-percent-compared-to-placebo-for-patients-with-mild-or-moderat/   https://www.sciencemediacentre.org/expert-reaction-to-interim-analysis-of-oral-antiviral-molnupiravir/   #molnupiravir #CoVid #mutagenesis   

Join Katie as she infiltrates Ten Pines Wellness Center in search of a telepathic mutant held captive there.

Join Katie & Madame Pele in the next installment of her solo series, traveling coast to coast to find and rescue more mutants. 

Join Katie and her character Madame Pele in her solo series as she travels the world saving mutants; starting with saving a frigid, frozen Brazil.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.31.363515v1?rss=1 Authors: Hamano, F., Matoba, K., Hashidate, T., Suzuki, T., Miura, K., Hishikawa, D., Harayama, T., Yuki, K., Kita, Y., Noda, N. N., Shimizu, T., Shindou, H. Abstract: Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions and promotes several pathological conditions, including anaphylaxis and neuropathic pain. PAF is biosynthesized by two types of lyso-PAF acetyltransferases: lysophosphatidylcholine acyltransferase 1 (LPCAT1) and LPCAT2, which are constitutive and inducible forms of lyso-PAF acetyltransferase, respectively. Because LPCAT2 mainly produces PAF under inflammatory conditions, understanding the structure of LPCAT2 is important for developing specific drugs against PAF-related inflammatory diseases. Although the structure of LPCAT2 has not been determined, the crystal structure was reported for Thermotoga maritimaPlsC, an enzyme in the same enzyme family as LPCAT2. In this study, we identified residues in mouse LPCAT2 essential for its enzymatic activity and a potential acyl-coenzyme A (CoA)-binding pocket, based on homology modeling of mouse LPCAT2 with PlsC. We also found that Ala115 of mouse LPCAT2 was important for acyl-CoA selectivity. In conclusion, these results predict the structure of mouse LPCAT2. Our findings have implications for the future development of new drugs against PAF-related diseases. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.31.363309v1?rss=1 Authors: Weng, Y. L., Naik, S. R., Dingelstad, N., Kalyaanamoorthy, S., Ganesan, A. Abstract: The 2019 novel coronavirus pandemic caused by SARS-CoV-2 remains a serious health threat to humans and a number of countries are already in the middle of the second wave of infection. There is an urgent need to develop therapeutics against this deadly virus. Recent scientific evidences have suggested that the main protease (Mpro) enzyme in SARS-CoV-2 can be an ideal drug target due to its crucial role in the viral replication and transcription processes. Therefore, there are ongoing research efforts to identify drug candidates against SARS-CoV-2 Mpro that resulted in hundreds of X-ray crystal structures of ligand bound Mpro complexes in the protein data bank (PDB) that describe structural details of different chemotypes of fragments binding within different sites in Mpro. In this work, we perform rigorous molecular dynamics (MD) simulation of 62 reversible ligand-Mpro complexes in the PDB to gain mechanistic insights about their interactions at atomic level. Using a total of ~2.25 microseconds long MD trajectories, we identified and characterized different pockets and their conformational dynamics in the apo Mpro structure. Later, using the published PDB structures, we analyzed the dynamic interactions and binding affinity of small ligands within those pockets. Our results identified the key residues that stabilize the ligands in the catalytic sites and other pockets in Mpro. Our analyses unraveled the role of a lateral pocket in the catalytic site in Mpro that is critical for enhancing the ligand binding to the enzyme. We also highlighted the important contribution from HIS163 in this lateral pocket towards ligand binding and affinity against Mpro through computational mutation analyses. Further, we revealed the effects of explicit water molecules and Mpro dimerization in the ligand association with the target. Thus, comprehensive molecular level insights gained from this work can be useful to identify or design potent small molecule inhibitors against SARS-CoV-2 Mpro. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.13.337147v1?rss=1 Authors: Nair, S., Shrikumar, A., Kundaje, A. Abstract: Deep learning models such as convolutional neural networks are able to accurately map biological sequences to associated functional readouts and properties by learning predictive de novo representations. In-silico saturation mutagenesis (ISM) is a popular feature attribution technique for inferring contributions of all characters in an input sequence to the model's predicted output. The main drawback of ISM is its runtime, as it involves multiple forward propagations of all possible mutations of each character in the input sequence through the trained model to predict the effects on the output. We present fastISM, an algorithm that speeds up ISM by a factor of over 10x for commonly used convolutional neural network architectures. fastISM is based on the observations that the majority of computation in ISM is spent in convolutional layers, and a single mutation only disrupts a limited region of intermediate layers, rendering most computation redundant. fastISM reduces the gap between backpropagation-based feature attribution methods and ISM. It far surpasses the runtime of backpropagation-based methods on multi-output architectures, making it feasible to run ISM on a large number of sequences. An easy-to-use Keras/TensorFlow 2 implementation of fastISM is available at https://github.com/kundajelab/fastISM, and a hands-on tutorial at https://colab.research.google.com/github/kundajelab/fastISM/blob/master/notebooks/colab/DeepSEA.ipynb. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.04.283424v1?rss=1 Authors: Sun, H., Liu, F., Ye, Z., Baker, A. P., Zwiebel, L. J. Abstract: Mosquitoes rely heavily on their olfactory systems for host seeking, selection of oviposition sites, and avoiding predators and other environmental dangers. Of these behaviors, the preferential selection of a human blood-meal host drives the vectorial capacity of anthropophilic female Anopheles coluzzii mosquitoes. Olfactory receptor neurons (ORNs) are dispersed across several appendages on the head and express an obligate odorant receptor co-receptor (Orco) coupled with a tuning odorant receptor (OR) to form heteromeric, odor-gated ion channels in the membrane of these neurons. To examine the mechanistic and functional contributions of Orco/OR complexes to the chemosensory processes of An. coluzzii, we utilized CRISPR/Cas9 gene editing to create a line of homozygous, Orco-knockout, mutant mosquitoes. As expected, orco-/- ORNs across both adult and larval stages of An. coluzzii display significantly lower background activity and lack nearly all odor-evoked responses. In addition, blood-meal-seeking, adult female, orco-/- mutant mosquitoes exhibit severely reduced attraction to human- and non-human-derived odors while gravid females are significantly less responsive to established oviposition attractants. These results reinforce observations in other insects that Orco is crucial in maintaining the activity of ORNs. In that light, it significantly influences a range of olfactory-driven behaviors central to the anthropophilic host preference that is critical to the vectorial capacity of An. coluzzii as a primary vector for human malaria. Copy rights belong to original authors. Visit the link for more info

Dr Michael Dent shares some of the research from the brand new IDTechEx report, "Genetic Technologies in Agriculture 2020-2030".For more information on this report, please visit www.IDTechEx.com/GeneticAgriThe report assesses the following:- Selective breeding and computational strategies used to improve efficacy- Mutagenesis strategies- Genetically modified organisms (GMOs): transgenics and cisgenics- Genome editing: CRISPR, TALENs and ZFNs- CRISPR: IP issues and potential consequences- Synthetic biology in crop agriculture- The global regulatory landscape- Consumer factors in the uptake of genetic technologies in agriculture- The future of genetic technologies in agriculture: 10-year forecasts by technology and by region The report is based on extensive research into the sector, including analysis and data from over 20 companies, including Bayer (including Monsanto), BASF, Syngenta (ChemChina), Corteva Agriscience, Calyxt, Ginkgo Bioworks, Pivot Bio, and AgBiome.For more information on this report, please visit www.IDTechEx.com/GeneticAgri or for the full portfolio related research available from IDTechEx please visit www.IDTechEx.com/Research.IDTechEx guides your strategic business decisions through its Research, Consultancy and Event products, helping you profit from emerging technologies. For more information on IDTechEx Research and Consultancy, contact research@IDTechEx.com or visit www.IDTechEx.com.

Keeping global temperatures to safe levels will require unlikely and disruptive discoveries from unexpected places. Medicine is a promising frontier. Kin Chan discusses his medical research on DNA damage and cancer, and explains how an enzyme that he discovered could eventually help pave the way for a new generation of low-carbon fuels. Recommendations: Mechanisms of Mutagenesis by Kin Chan

Terra AD's jolliest holiday is here at last! Join the judges for the Feast of the Mutagenesis and exchange radioactive gifts with the mutants in your life.

In this episode of The Vitality Secret Podcast, friend and anti-ageing expert and enthusiast, Nic Burns, shares how she reversed Lupus. This is loaded with content, covering the physical, mental, emotional and spiritual pillars of Vitality.  SHOW NOTES 2.0 - Pain developed was as young as 3, “winter pains” 3.0 – Was told it was growing pains 4.0 – Lots of different symptoms, rheumatologist, ‘spider bite' 5.0 – Diagnosis with Lupus, a big shock for Nic for personal reasons 6.0 - Friend with Lupus was on 25 drugs a day and died at 28 7.0 – Was advised to take medication 8.0 – Was already researching anti-ageing & applied the same principles 9.0 – Main foods that she removed to experience changes 10.0 – Mutagenesis of wheat 11.0 – Tiredness, kids, stress (plus learning about Lupus), pretended to take the medication 12.0 – After 12 months, went for a check up, “stable”  13.0 – Blood tests now show no evidence of Lupus in blood 14.0 – “No doctors are educated in nutrition” 15.0 – Nic's journey was longer due to slowly learning. Now the knowledge is accessible fast 16.0 – Mention of Radical Remission for anyone with cancer, get the diagnosis from Western Medicine, then seek out ways to heal yourself 17.0 – Nic inspired her doctor to turn to Functional Medicine! 18.0 – The history of the medical industry & mind-body work 19.0 – Louise Hay “Lupus is a sign of giving up on life,” mindset & positivity 20.0 – Nic was helped with visualisation, “you can heal your life with your mind,” hypnotherapy 21.0 – Quantum physics; Dr Joe Dispenza, Gregg Braden, Dr Bruce Lipton 22.0 – Commanding your cells to heal 23.0 – The placebo effect vs the nocebo effect 24.0 – Using your mind to heal – Physical, mental & emotional pillars of Vitality 25.0 – The focus of gut health & healing – ‘face-gut connection' & leaky gut 26.0 – “Heal your gut, heal your body” 27.0 – Nutrition is huge, and not the only place. It's the first place to look. 28.0 – Immediate response to gluten consumption & other foods 29.0 – Wahl's protocol and Dr Amy Myers 30.0 – Why certain vegetables create gut challenges. Nature is our medicine. 31.0 – The ketogenic diet 32.0 – “Butter on your coffee? You dirty animal!” 33.0 – Muscle testing 34.0 – Vaccines 35.0 – Staggering vaccines in a safer way 36.0 – Is watching the news a good thing? 38.0 – Clean eating, anti-aging coffee! 39.0 – Staggering an anti-inflammatory lifestyle 40.0 – Physical, movement, vibrating 41.0 – The vibration plate – bone, immune system 42.0 – Vibration plate releases toxins – great for the lazy person – an awesome biohack! 43.0 – Grounding, sauna, far & near infrared. Swedish Sauna. HIIT 44.0 – Benefits of HIIT – max 20 minutes 45.0 – Our muscles and generation of electricity 47.0 – “You can't explain to just change one thing” – the holistic approach 48.0 – Nic's Serum business – her passion is anti-aging and facial acupuncture 49.0 – Tightening the skin, reducing fine lines and wrinkles, smell to stimulate brain 50.0 – Feel good, look good, smell good – www.njbageless.co.uk 51.0 – What is Nic's #1 Vitality Secret? 52.0 – Choosing the cleanest drink – ‘drink hack' 53.0 – IMPACT!! Let's share this with the world. 54.0 – The body is an amazing self-healing machine!!   Nic's Anti-ageing serum website: http://njbageless.co.uk Ignite Your Inner Healer: https://VitalitySecret.com/Ignite If you'd my support, go here: https://VitalitySecret.com/Coaching  

Nels and Vincent reveal a highly conserved protein that acts as an evolvability factor, increasing mutation and the ability of bacteria to become resistant to antibiotics. Hosts: Nels Elde and Vincent Racaniello Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiEVO Inhibiting evolution of antibiotic resistance (Mol Cell) Pushing viruses over the error threshold (virology blog) Image credit Letters read on TWiEVO 38 Science Picks Nels- Science/nature illustrator/artist Rachel Ignotofsky (website, Etsy shop) Vincent- Life Ascending by Nick Lane  Music on TWiEVO is performed by Trampled by Turtles Send your evolution questions and comments to twievo@microbe.tv

The TWiVumvirate reviews this years crop of Nobel Prizes, and how cells prevent leakage of mitochondrial double-stranded RNA into the cytoplasm, which would otherwise lead to the production of interferon. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Sea Phages program and application materials Plant biologists penalized by CNRS (The Scientist) 2018 Nobel Prize in Medicine (pdf) 2018 Nobel Prize in Chemistry (pdf) 2018 Nobel Prize in Physics (pdf) 2018 Nobel Peace Prize (Nobel) Mitochondrial dsRNA triggers IFN (Nature) Letters read on TWiV 514 Timestamps by Jolene. Thanks! Weekly Science Picks 1:28:33 Alan- Compound Interest chemistry graphics Kathy- Writing letters of recommendation Guidelines Trix & Psenka Schmader et al. Madera et al. Dickson- Japan Fireworks Vincent - The Game of T-Cells and Apple Park Lego Listener Picks Steve - Shomu's Biology Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Judge James and Judge Forrest are joined by Judge Marc Plourde. Of course, this means another extra long Mutagenesis and an extra long episode! Join us for our deep dive on the Healer and Shaman classes. Wander the wastelands with us a while!

Judge Forrest and Judge James are joined by two very, very special guests in this, Glowburn's 12th episode. We explore character generation and offer twice the fun of the Mutagenesis! More rads than you can absorb in one episode and still remain un-mutated!

Dr Hilkens speaks with ecancer at the 1st Cancer Research @ Bath Symposium about identifying mutational pathways that led to breast cancer. He focuses on the role of spondins, which have been implicated in tumourigenesis in colon cancer, as driving stem cell progression to cancer, and explains his methods of insertional mutagenesis to identify 30 potential oncogenes. Dr Hilkens' research on one of the genes, insulin receptor substrate (IRS4), has been recently published in Molecular and Cellular Oncology, and he also considers the role of LTR6 in stem cell behaviour.

Judge Forrest and Judge James speak AT LENGTH with Judge Brendan LaSalle about MCC, X-crawl, running, writing, and playing games. Blade Runner weaponry and the Hofstader Cube feature in Mutagenesis, while Judge Brendan creates some cool stuff on the fly. Hear the man in action! Then run, run as fast as you can, running man!

Like purple Zeeth grass blooming after a nuclear winter, Glowburn is back! This time, our Great Disaster centers on our robot masters . . . er, robot servants! There are changes in the HQ bunker, discussions of topics as varied as robot PC campaigns and Van Hagar, and of course, voices of the Ancient Ones (for real!). Mutagenesis highlights include a non-lethal, but very annoying robot for suppressing mutant uprisings and pointers on reskins for the character classes you already know and love. Opening and closing theme song is Juno by Chronox.

Dr Harris talks to ecancertv at SABCS 2015, about his research on APOBEC3B and its correlation to drug resistance in breast cancer. APOBEC, particularly APOBEC3B is a brand new source of mutation in cancer, he observes. Infection with the human papillomavirus (HPV) has been shown to account for upregulation of APOBEC3B in cervical and head/ and neck cancers but it is not clear how it becomes changed in on-viral malignancies such as breast cancer. Dr Harris discusses data that show that upregulation in breast cancer may occur via the PKC-NFκB pathway. These data establish the first mechanistic link between APOBEC3B and a common signal transduction pathway, he says, suggesting that using inhibitors that target this pathway may be of benefit.

Hosts: Vincent Racaniello, Michael Schmidt, and Elio Schaechter. Guest: Harris Wang Harris joins Vincent, Elio, and Michael to describe multiplex automated genome engineering, a method for targeting many modifications in a population of bacterial cells. Subscribe to TWiM (free) on iTunes, Stitcher, Android, RSS, or by email. You can also listen on your mobile device with the Microbeworld app. Links for this episode  MAGE - Multiplex automated genome engineering (Nature) Genome-scale promoter engineering (Nat Methods) Manipulating microbial communities in situ (Trends Genet) Engineering human microbiomes (Meth Mol Biol) Genome-scale engineering (Mol Syst Biol) Economic framework of microbial trade (PLoS One) Tardigrade t-shirt Image credit This episode is sponsored by Microbe Magazine Podcast and ASM Microbe 2016 Music used on TWiM is composed and performed by Ronald Jenkees and used with permission. Send your microbiology questions and comments (email or mp3 file) to twim@twiv.tv.  

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler The TWiV teams reviews a MERS-coronavirus serosurvey and an outbreak in South Korea, and constraints on measles virus antigenic variation. Links for this episode Richard Elliott on TWiV 177 MERS-CoV outbreak, South Korea (ProMedMail) MERS-CoV serosurvey (Lancet Inf Dis) Biolabs in your backyard (USA Today) Constraints on measles virus antigenic variation (Cell Rep) Selection bias and bombers (John D. Cook) Virology (journal) Image credit: Cameron Moll Letters read on TWiV 340 Weekly Science Picks Dickson - Singapore Flower Dome  and Cloud ForestAlan - Rescuing biomedical researchKathy - The value of basic researchRich - Virology 60th Anniversary IssueVincent - Careers in virology: Science writing and journalism Listener Pick of the Week Pritesh - How T cells kill cancer cellsRamon - The power of herd immunity Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

James M. Gentile is the Dean for Natural and Applied Sciences at Hope College and the past President of Research Corporation for Science Advancement, a Tucson, AZ-based foundation dedicated to science since 1912. Jim received his Master's and PhD in Genetics from Illinois State University and conducted a Postdoctoral Fellowship at Yale University School of Medicine. Jim then joined the faculty of Hope College where he remained for nearly 30 years before becoming President of Research Corporation for Science Advancement, a foundation that funds innovative scientific research. In 2013, Jim returned to Hope College where he is today. Jim has received many awards and honors during his career, including the Alexander Hollaender Research Excellence Award from the Environmental Mutagen Society, the Cancer Medallion of the Japanese National Cancer Institute, and the Science Medal of Distinction of Pisa, Italy. He is a Fellow of the American Association for the Advancement of Science, a National Associate of the National Academies of Sciences, and a National Academies education mentor. Jim was also honored by Illinois State University with an Alumni Achievement Award and election to the university Hall of Fame, and was given a Special Achievement Award by the Council on Undergraduate Research. Jim is here with us today to tell us all about his journey through life and science.

Roger D. Cox, Medical Research Council, Harwell - UK speaks on "ENU mutagenesis 1: Aging screens". This seminar has been recorded at ICTP Trieste by ICGEB Trieste

Circadian clocks are endogenous cellular mechanisms that control daily rhythms of physiology and behaviour. The adjustment of the circadian clock to the 24 h period of a day is commonly accomplished by several environmental cues, e.g. temperature, light and nutrition. For one light input pathway the mechanism that synchronises or entrains Neurospora’s clock is supposed to be known. Nevertheless, there are plenty more environmental cues that have an obvious impact on the circadian clock, e.g. temperature. The environmental cue “temperature” was underrepresented in studies about Neurospora’s circadian clock, while several clock studies focused on stationary conditions rather than changing ones. As a result, the functionality and adaptation of circadian clocks were underestimated, and thus new clock components could be overlooked due to screening on constant darkness. It was therefore important to develop a novel strategy in screening mutants that challenged the circadian clock of Neurospora crassa entirely on temperature alternations. A temperature cycle of low amplitude (22° C cold and 27° C warm) and of short period (8 h cold and 8 h warm) applied as Zeitgeber stimulus. A mutant library was created with insertional mutagenesis via electroporation in order to transform a BASTA resistance gene into conidial nuclei. As a consequence, a novel method of rescuing the mutations was established, which combined the process of mapping, partial cloning and PCR and was called Size Selected Fragment Plasmid Rescue or SSFPR in short. During the screening of several hundred mutants among the novel protocol, a known clock gene, frequency, was identified and characterised. The identification of several mutants with altered clock phenotypes has on one hand confirmed the general approach of this study and on the other proved that the greater sensitivity for the temperature screen can bee used to detect mutant phenotypes.

Episode 5 of Privateer Press Primecast has arrived! Join co-hosts Simon Berman and Will Shick as they interview fellow staffers at Privateer HQ. Development Manager, David “DC” Carl talks with Will about WARMACHINE: Vengeance, how it was developed, and what it offers gamers. Next, Simon interviews Skull Island eXpeditions author, Orrin Grey, about his novella, Mutagenesis. Marketing Manager, Lyle Lowery has lots to say about Zombies Keep Out and Privateer Play Day. Doug Seacat returns to Primecast to delve deep into the story and characters of WARMACHINE: Vengeance and drops a dire hint to the future of western Immoren. 

Hosts: Vincent Racaniello and Dickson Despommier Vincent and Dickson meet with Judith Straimer and Marcus Lee to discuss their method for site-specific genome editing in Plasmodium falciparumusing zinc finger nucleases. Right-click to download TWiP #46 (46 MB .mp3, 64 minutes). Links for this episode: Judith Straimer and Marcus Lee Genome editing in P. falciparum with zinc finger nucleases (Nature Methods) Zinc finger nucleases (Sigma-Aldrich) Gene-editing nucleases (Nature Methods) Illustration by Andrew Lee Contact Send your questions and comments (email or mp3 file) to twip@twiv.tv.

Sat, 13 Feb 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13029/ https://edoc.ub.uni-muenchen.de/13029/1/Buerck_Lelia_van.pdf Bürck, Lelia van

Wed, 27 Jan 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11284/ https://edoc.ub.uni-muenchen.de/11284/1/Damsma_Gerke.pdf Damsma, Gerke E. ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Synthetic PDZ domain variants reveal how this domain can evolve to produce proteins with different binding specificities.

According to recent epidemiological studies, primary aldosteronism is considered to be responsible for almost 10-15% of all cases of arterial hypertension. The genetic background of this common disease, however, has been elucidated only for the rare familial types whereas in the large majority of sporadic cases genetic causes or modifiers still remain unclear. In an attempt to define novel genetic mechanisms of hyperaldosteronism we utilized a random mutagenesis screen after treatment with the alkylating agent N-ethylnitrosourea (ENU) and phenotypically characterized affected mice for their blood aldosterone levels. As the detection method we used a time resolved fluorescent immunoassay which allows the measurement of aldosterone in very small murine plasma volumes. Using this assay we determined the normal aldosterone values for C3HeB/FeJ wild type mice under baseline conditions and following specific stimulation and suppression tests. We could demonstrate the expected increase in aldosterone response upon ACTH stimulation, a high potassium diet and an angiotensin II injection, as well as the decrease after a dexamethasone suppression test, a normal saline load test and a fludrocortisone suppression test. On the molecular level, the expression of aldosterone synthase showed a similar pattern with a fast response to the investigated stimuli. These tests should later be applied to the mouse lines derived from the ENU screen, in order to investigate potential abnormal response to these stimuli in comparison to wild type animals. Furthermore, aldosterone measurement was carried out in more than 2000 F1 offspring (of both genders) of chemically mutated inbred C3HeB/FeJ mice. From these tested F1 offspring, aldosterone levels were consistently elevated (defined as levels above 3 SD over the mean of untreated animals)upon repeated measurement in 9 animals (8 females and 1 male). Further breeding of affected female animals gave rise to F2 pedigrees from which four established lines displayed high aldosterone values. These animals served for a detailed phenotypic characterization and showed an increased aldosterone to renin ratio, low potassium values and normal renal function in line with the presence of primary aldosteronism. In addition, the investigation of their cardiac phenotype showed increased collagen deposits and subsequently cardiac fibrosis, as also observed in patients suffering from primary aldosteronism. In the future, genetic SNP analysis can be performed to identify underlying genetic loci, responsible for this trait. Taken together, these data demonstrate the feasibility of a phenotype-driven mutagenesis screen to detect and establish mutant mouse lines with a high aldosterone phenotype.

Background: Clinical chemical blood analysis including plasma electrolytes is routinely carried out for the diagnosis of various organ diseases. Phenotype-driven N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects used plasma electrolytes as parameters for the generation of novel animal models for human diseases. Methods: Here, we retrospectively evaluated the use of the plasma electrolytes calcium, chloride, inorganic phosphorus, potassium and sodium in the Munich ENU mouse mutagenesis project where clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in various plasma parameter levels. Results: We identified a small number of animals consistently exhibiting altered plasma electrolyte values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters calcium and potassium. Published data from other phenotype-driven ENU projects also included only a small number of mutant lines which were generated according to altered plasma electrolyte levels. Conclusion: Thus, use of plasma electrolytes detected few mouse mutants in ENU projects compared to other clinical chemical blood parameters.

The natural design of the photosystems of plants and photosynthetic bacteria using chlorophylls (Chls) or bacteriochlorophylls (BChls) as photoreceptors are robust. The basic principles of the biological system of light-harvesting complex 2 (LH2) are studied with the use of natural and model sequences expressed in vivo in modified Rhodobacter (Rb) sphaeroides strains. Three aspects have been explored in the thesis: (1) BChl’s macrocycle-protein interactions, (2) BChl’s phytol-protein interactions underlying the structural and functional assembly of the pigment-protein complexes, and (3) LH2-lipid interactions and the role of these interactions in photosynthetic membrane morphogenesis. BChls’ macrocycle-protein interactions: Residues at the immediate BChl-B850/protein interface are found to have little effect on specifying the BChl-B850 array, and their light-harvesting activity in LH2. Nevertheless, these residues are important for the structural thermal stability. With the use of ‘rescue’ mutagenesis of the model BChl binding site, the hydrogen-bond between αSer -4 and the C131 keto carbonyl group of βBChl-B850 is shown to be a crucial motif for driving the assembly of model LH2 complex. Possibilities for residue modifications are limited in the β-subunits as compared to the α-subunits, which suggests that the two polypeptides have distinct roles in complex assembly. In the β-subunits, there are residues detected adjacent to the BChl-B850 site which are critical for the assembly of LH2. BChls’ phytol-protein interactions: Mutagenesis of residues closely interacting with the BChl-B850 phytol moiety result in the pronounced loss of BChl-B800 from LH2. Dephytylation of bound BChls within assembled LH2 to BChlides also resulted in the loss of BChl-B800 and destabilisation of LH2 structural assembly. Thus, the phytol chains were shown to be important for optimal pigment binding, especially for BChl-B800; which appears to be highly sensitive to the proper packing of the phytols. The pattern of phytol interactions with their surrounding environments are significantly different for α- and β-ligated (B)Chls. The phytols of β-ligated (B)Chls, as opposed to α-ligated (B)Chls, have ample and specific interactions with residues of the binding helix which may contribute to the tertiary interactions of helices. LH2-lipids interactions: Phospholipid determination of LH2 only expressing strains of Rb sphaeroides shows that the nonbilayer-forming phospholipid, phosphatidylethanolamine (PE) is present in elevated amounts in the intracytoplasmic membranes and in the immediate vicinity of the LH2 complex. In combination with βGlu -20 residue and the carotenoid headgroup at the N-terminus of the transmembrane β-helices is shown to influence the composition of lipids surrounding LH2. Specific local interactions between LH2 protein and lipids not only promote LH2 protein stability but appear to modulate the morphology of intracytoplasmic membranes. Based on these findings, the presence of LH2-lipid specificity is postulated. The approach of using model αβ-sequences with simplified pigment binding sites allows us to study the underlying factors involved in LH2 assembly and function. This gives rise to a better understanding of the interplay between BChl, apoproteins and membrane lipids in the assembly of a highly efficient light-harvesting complex in its native lipid-environment.

Toxic reactions with the molecules of life.

Lecture Slides in PDF format

Toxic reactions with the molecules of life.

Lecture Slides in PDF format

Background: The ENU Mouse Mutagenesis Project aims at a large-scale, systematic production of mouse mutants using the alkylating agent ethyl-nitrosourea (ENU). Offspring of mutagenized mice are subjected to a multiparameter screen to detect alterations in various phenotypes with the ultimate goal of identifying novel genes relevant for the expression of the phenotype. Using this approach, we have analyzed plasma IgE concentrations to identify mouse mutants with aberrant plasma IgE levels. Methods and Results: ENU-mutagenized male C3HeB/FeJ were mated to wild-type females to produce F1 offspring. F1 animals were analyzed for alterations in their plasma IgE concentrations that showed a dominant mode of inheritance, or bred further to screen for recessive phenotypes. Plasma IgE concentrations were deter mined by ELISA and a normal range for plasma IgE was established using C3HeB/FeJ wild-type animals. So far we have tested 6568 F1 animals. Repeated testing confirmed a stable aberrant IgE phenotype in 124 animals. To confirm the genetic basis of the observed phenotype, these mice were subjected to confirmation crossing. Currently we have established 9 independent m uta nt mouse lines (3 with high plasma IgE and 6 with plasma IgE below detection limit) that have been genetically confirmed and additional 24 variant mouse lines are currently undergoing confirmation testing. Conclusion: ENU mouse mutagenesis allowed us to generate and identify mouse mutants with aberrant plasma IgE levels, which may be used to characterize novel genes involved in IgE regulation and may serve as animal models for IgE-mediated diseases. Copyright (C) 2001 S. Karger AG, Basel.

The function of the CD4 cell surface protein as coreceptor on T helper lymphocytes and as receptor for HIV makes this glycoprotein a prime target for an immune intervention with mAb. A detailed understanding of the structural determinants on the therapeutic CD4 mAb that are involved in Ag binding or are recognized by anti-idiotypic mAb (anti-Id) may be important for designing antibodies with optimal therapeutic efficacy. Seven anti-Id raised against the CD4 mAb M-T310 were selected from a large panel with the intention to obtain CD4 mimicking structures with specificity foHr IV gp120. The selected anti-Id did not reacwt ith other CDCspecific mAb cross-blocking M-T310. Among these, mAb MT404, although having the same L chain as M-T310 and a VH region sequence differing onlya t 14 amino acid positions, was not recognized by the anti-Id. MT310 H chain complexed with the J558L L chain reacted with all anti-Id, thus demonstrating that the recognized idiotopes are located within the VH region. To identify the idiotopes of M-T310 seen by the anti-Id, variants of M-T404 containing one or more of the M-T3 1 O-derived substitutions were generated by oligonucleotide-directed mutagenesis. The reactivity pattern of the mutant proteins with the anti-Id demonstrated that the idiotopes reside within the complementarity determining region (CDR) 2 and CDR3 loops of the VH region. A major idiotope was definebdy a single amino acid in CDR2 that was recognized by three anti-Id, whereas the four other anti-Id reacted with determinants of CDR3. Although the performed amino acid substitutions did influence the Id recognition, Ag binding was not significantly affected, suggesting that none of the anti-Id can be considered as a mimicry of the CD4 Ag

Mon, 1 Jan 1990 12:00:00 +0100 https://epub.ub.uni-muenchen.de/3755/1/3755.pdf Oesterhelt, Dieter; Zinth, Wolfgang; Lauterwasser, Christoph; Holzapfel, Wolfgang; Finkele, Ulrich; Stilz, Hans Ulrich

The chicken lysozyme gene is constitutively expressed in macrophages, in oviduct cells its expression is controlled by steroid hormones, and in fibroblasts the gene is not expressed. A fusion gene consisting of promoter sequences of the lysozyme gene from –208 to +15 in front of the chloramphenicol acetyltransferase (CAT) coding region was more than 50 times less active in non-expressing cells as compared to expressing cells. In order to identify the element(s) responsible for this cell-type specificity 31 different linker scanning mutations were generated within this promoter fragment and analyzed by transient transfections in the three types of chicken cells mentioned above. Three mutation sensitive regions located around position –25, –100 and between –158 and –208 were detected in each cell type, however, several LS mutations displayed clear cell-type specific differences in their phenotypic effects. Interestingly, a few LS mutations led to an increase in promoter activity in fibroblasts suggesting that the corresponding wildtype sequences represent binding sites for negatively acting transcription factors.